Lignocellulosic biomass is characterized by a complex structure consisting of three main polymers: cellulose, hemicellulose and lignin. The cellulose and possibly the hemicelluloses are the targets of enzymatic hydrolysis, but they are not directly accessible to enzymes.
These substrates therefore have to undergo a pretreatment prior to the enzymatic hydrolysis stage. The pretreatment aims to modify the physical and physico-chemical properties of the lignocellulosic material in order to improve the accessibility of the cellulose trapped in the lignin and hemicellulose matrix. These pretreatments can be of different types: acidic boiling, alkaline boiling, steam explosion or Organosolv processes can be mentioned.
The enzymatic hydrolysis stage allows cellulose and hemicelluloses to be converted to sugars using cellulolytic and/or hemicellulolytic enzymes. Microorganisms such as fungi belonging to the Trichoderma, Aspergillus, Penicillium or Schizophyllum genera, or anaerobic bacteria belonging for example to the Clostridium genus, produce these enzymes containing notably cellulases and hemicellulases, suited for total hydrolysis of the cellulose and of the hemicelluloses.
The sugars obtained by lignocellulosic biomass hydrolysis are pentoses (mainly xylose and arabinose), disaccharides (cellobiose) and glucose that can be fermented by microorganisms. Glucose can for example be readily converted to ethanol by the Saccharomyces cerevisiae yeast during the alcoholic fermentation stage.
Finally, a distillation stage allows to separate and to recover the fermentation product thus obtained, ethanol in the previous case, from the fermentation must.
Various technico-economic studies show the necessity of reducing the cost linked with the enzymatic hydrolysis stage in order to bring the cost of the ethanol produced to values close to the ethanol obtained from starch.
One means of decreasing the costs consists in optimizing the operating conditions of the cellulase production method so as to increase productivity or to obtain an enzymatic cocktail having an improved specific activity.
The most commonly used microorganism for cellulase production is the filamentous Trichoderma reesei fungus. The wild strains have the ability to secrete, in the presence of an inductive substrate such as cellulose or lactose for example, an enzymatic complex suited for cellulose hydrolysis. The enzymes of the enzymatic complex comprise three major types of activities: endoglucanases, exoglucanases and cellobiases.
The complex cellulase synthesis regulation mechanisms require particular implementations for their production in a reactor. Using strains that are not sensitive to catabolic repression and soluble inductive substrates usable on a large scale, such as lactose, has allowed to obtain significant productions of the order of 40 g/L extracellular proteins by T. reesei CL 847 according to a method described in Bioresource Technol. (1992) 39, 125-130. In this method, fermentation takes place in two stages, a first stage of batch production of T. reesei cells and a second stage of fed batch supply of the inducer at a rate preventing accumulation thereof in the medium.
The physiological state of the microorganism being not always maintained at its optimum level for enzyme production, phenomena of growth of nonproductive cells, or of unwanted cell lysis or sporulation leading to an enzyme productivity decrease, are observed in some cases. The balance between optimum production states and unwanted physiological states is rather sensitive. Bailey et al. (Appl. Microbiol. Biotechnol. 2003 62: 156-162) have shown that the productivity of cellulases is optimum when the cells are in a state close to the carbon-containing substrate limitation in the medium. A method of adding carbon-containing substrate using monitoring the basic consumption rate used for pH control has been proposed.
Furthermore, the method described in patent EP-B1-0,448,430 requires an optimized sugar supply rate of 35 to 45 mg·g−1·h−1. In fact, too high a supply rate causes an accumulation of carbon-containing substrate in the medium, which leads to a metabolic change towards biomass production instead of enzyme production. On the other hand, too low a supply rate leads to biomass lysis and to production loss. Furthermore, Trichoderma reesei excretes then more proteases reconsuming part of the cellulases produced.
The present invention provides a method allowing to maintain the microorganism in the state corresponding to its optimum level for enzyme production.